Project Abstract S-adenosyl-L-methionine (AdoMet) is a common methyl donor in cellular epigenetic modification of macromolecules to regulate gene expression. Methionine adenosyltransferase (MAT) is the sole enzyme responsible for AdoMet synthesis from ATP and methionine substrates. Three MAT isozymes are found in humans, with only the MAT2A isoform being expressed in most tissues and cancer cells. Over expression of MAT2A in cancer cells has been noted for some time. Recent studies have shown that a common gene deletion in cancer cells of 5?-methylthioadenosine phosphorylase (MTAP) creates susceptibility to MAT2A inhibition due to increased cellular dependence on AdoMet synthesis. Our lab has previously targeted MTAP using tight-binding transition state analogues that were successful in reducing cancer cell growth in cell culture and mouse xenografts. Cancer cells conditioned to MTAP inhibitor resistance were analyzed and a single gene amplification event at the MAT2A gene locus was identified. In this proposal, we will use two powerful methods of enzyme inhibitor design, click chemistry and transition state analogues, to target MAT2A. These innovative chemistry approaches have been applied to other enzyme targets to generate some of the tightest binding inhibitors known. Click chemistry and transition state analogue approaches allow for creation of bisubstrate analogues that target both the ATP and methionine binding sites of MAT2A. This simultaneous targeting of two enzyme active site groups increases the inhibitor affinity, as it resembles the short-lived but high-affinity intermediate reaction species. Inhibitors generated through these methods will be subject to structural, thermodynamic, and kinetic analysis. Efficacy of these high affinity MAT2A inhibitors on reducing cancer cell growth and viability will be analyzed on MTAP-/- and MTAP+/+ cancer cell lines. Co-treatment of cancer cell lines with MTAP transition state analogues will explore synergistic effects of MTAP and MAT2A. We hypothesize that MAT2A inhibitors generated from this work will amplify the anti-cancer effects MTAP inhibitors and provide a novel treatment for MTAP-/- cancers.